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. 2017 Dec 4;7:16892. doi: 10.1038/s41598-017-16928-8

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Antigen-specific cellular and humoral immunity induced by CLDN6-presenting MV. (a) Schematic depiction of immunization experiments. (b) IFN-γ ELISpot analysis using splenocytes of vaccinated mice with indicated viruses expressing CLDN6 or MLV-gag. Viruses expressing VLPs, but no antigen (MV_vac2-gag) served as negative controls. Splenocytes isolated 21 days after boost immunization and analyzed for IFN-γ-production after incubation with ConA, viral antigen (MV bulk), specific or unspecific peptides (CLDN6 or ISQ, respectively), or without stimulation (mock). (c) Virus neutralizing titers (VNT) of with indicated viruses vaccinated animals’ sera were analyzed for neutralization of MV. LLOD = VNT 20. (d) Flow cytometry histograms of CLDN6-expressing CT26 cells stained with sera of representative mice vaccinated with indicated viruses before (solid black line), after prime (solid grey line), or after booster (filled grey area) vaccination. Staining with anti-CLDN6 mAb served as positive control (e) Analysis of CLDN6-specific antibody concentration in serum samples of vaccinated mice determined by FACS as shown in panel D according to MFI of stained cell populations. Dots represent single animals (n = 5–6); horizontal line represents mean per group. Statistical differences between groups were assessed by Mann-Whitney test (b) or two-way ANOVA (d); *P < 0.05; **P < 0.01; ****P < 0.0001.